Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Abstract

Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTMtargeting antibodies have reported frequent nonspecific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phospho-threonine 231 (pT231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinitymatured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved > 20-fold over that of the wild-type antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity towards the nonphosphorylated target site, but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phospho-tau antibody. In cell-and tissueimaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTMspecific antibodies, and highlight the importance of assessing the specificity in the antibody engineering process.

High specificity of widely used phospho-tau antibodies validated using a quantitative whole-cell based assay

“…Site-specific phosphorylation of tau is critical in understanding the molecular biology of tau and is a welldefined target in neurodegeneration. The specificity of phospho-tau antibodies is critical for reproducible and sensitive detection (Li et al 2018;Lothrop et al 2013). Phospho-tau antibodies should interact only with the target epitope sequence with site-specific phosphorylation, and not with nonphospho-tau or phospho-tau at off-target sites.…”

“…1a) for its ability to discern phospho-specificity using 3 previously characterized anti-tau antibodies. Tau-5 is a pan-tau antibody that shows phosphorylation-independent binding (LoPresti et al 1995;Carmel et al 1996), 3.24 is a high specificity scFv to pThr231 tau (Li et al 2018), and ab131354 targets pSer262 tau but have shown cross-reactivity to non-phosphorylated tau (Ercan et al 2017). Tau-5 indeed showed binding to both EGFP-tau WT and iRFP-tau WT treated with λ PP (Fig.…”

“…Tau-5 (Tau210-241, Invitrogen # AHB0042, AB_2536235) is a monoclonal antibody recognizing phosphorylated and non-phosphorylated tau isoforms. Purified single-chain antibody variable fragment (scFv) 3.24 targeting pThr231 was reported previously (Li et al 2018).…”

High-specificity antibodies and detection methods for quantifying phosphorylated tau from clinical samples

“…1b ) [ 32 ]. The fact that these antibodies were raised using phosphorylated peptides as antigens illustrates the importance of performing negative selections to remove nonselective binders during the antibody screening process [ 33 , 34 ]. The structural analyses revealed that the antibodies engage the phosphate group using diverse complementarity determining region (CDR) residues including those in CDR H1, H2, H3, or L1 ( Fig.…”

CAR-T Therapy for Solid Tumors: Development of New Strategies

“…It is important to note that fine-tuning scFV to harbor high or low binding affinity is an important avenue for improving CAR-T cells in providing safer and better anti-tumor effect. Various protein engineering approaches such as directed evolution [84] , domain exchange [85] , coupled with high-throughput analysis using phage display [88] can assist in scFv fine tuning.…”